Preparation of monoclonal antibody against recombinant VacA of H.pylori

Objective To prepare monoclonal antibody(mAb) against vacuolating cytotoxin antigen(VacA) of H.pylori.Methods Recombinant VacA protein was expressed by genetically engineered strain of E.coli DH5α/pQE30-v,and then was purified by Ni2+-NTA affinity chromatography.The antigenicity of the recombinant protein was confirmed by Western blotting.Rabbits were immunized with the recombinant protein while the VacA antibody level in the rabbits was detected for confirmed the immunogenicity by ELISA.The splenocytes of the VacA protein-immunized mice were fused with myeloma cells Sp2/0.Hybridoma cells were selectively cultured by HAT medium and screened by indirect ELISA.The titer of monoclonal antibody secreted by hybridoma cells was detected,classified,and typed.Results Four hybridoma cell lines stably secreting specific mAbs against VacA were obtained and identified.Identification of subclass showed that mAbs were IgG2b,IgM,and IgG1,and all the light chains of mAbs were κ.Western blotting showed that IgG1mAb had a specific affinity for the recombinant VacA.Conclusion Four hybridoma cell lines secreting specific monoclonal antibody against VacA of Helicobacter pylori are established and the mAbs against VacA are successfully obtained,which might play an important role in preparing the VacA test kit and could afford data in exploring the mechanisms of the VacA cytotoxicity.